General term for a laboratory instrument used for deciphering the order of bases in a strand of DNA. Sanger sequencers detect fluorescence from different dyes that are used to identify the A, C, G, and T extension reactions. Contemporary or Pyrosequencer methods are based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.
URL | https://www.bco-dmo.org/dataset/781114 |
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Download URL | https://www.bco-dmo.org/dataset/781114/data/download |
Media Type | text/tab-separated-values |
Created | November 6, 2019 |
Modified | November 22, 2019 |
State | Final no updates expected |
Brief Description | 16S rRNA gene (from RNA) |
Acquisition Description
Sampling of cores was done in accordance with IODP protocols.
All C0014 samples were cored on 16 September 2010 via via HPCS (hydraulic piston coring system).
All C0015 samples were cored on 18 September 2010 via HPCS.
All C0017 samples were cored on 27 September 2010 via HPCS.
Core sections were kept frozen (-80°C) until DNA extractions. RNA was extracted using a phenol chloroform protocol. The RNA extract was DNAse treated using the Invitrogen™ Ambion™ Turbo DNA-free Kit and purified using the Qiagen® RNeasy Mini Kit. A cDNA library was made using the Qiagen ® QuantiTec Reverse Transcription Kit. Polymerase chain reactions used the KAPABiosystems® KAPA2G Robust HotStart ReadyMix PCR Kit for 32 cycles.
Sequencing of the product was performed on the Illumina® MiSeq 2500.
Processing Description
Data Processing: Demultiplexing was performed in Mothur (v.1.30.1).
Problem report: Upon analysis, the RNA dataset does not appear to reflect the same or similar information as the published 16S rRNA gene amplicon dataset. Rather, it indicates significant background noise from extraction and not an indigenous representation of the subsurface biosphere.
Instruments
The JOIDES Resolution's Advanced Piston Corer (APC) is used in soft ooze and sediments. The APC is a hydraulically actuated piston corer designed to recover relatively undisturbed samples from very soft to firm sediments.
More information is available from IODP (PDF).
Parameters
unique sample identification or number; any combination of alpha numeric characters; precise definition is file dependent
Latitude; positive values = North
latitude, in decimal degrees, North is positive, negative denotes South; Reported in some datasets as degrees, minutes
Longitude; positive values = East
longitude, in decimal degrees, East is positive, negative denotes West; Reported in some datsets as degrees, minutes
depth below seafloor. Includes mbsf (meters below seafloor) and cmbsf (centimeters below seafloor).
depth below seafloor. Includes mbsf (meters below seafloor) and cmbsf (centimeters below seafloor).
16S rRNA primer set
Dataset Maintainers
Name | Affiliation | Contact |
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Christopher H. House | Pennsylvania State University (PSU) | ✓ |
Leah D. Brandt | Pennsylvania State University (PSU) | ✓ |
Shannon Rauch | Pennsylvania State University (PSU) | |
Shannon Rauch | Pennsylvania State University (PSU) | |
Shannon Rauch | Woods Hole Oceanographic Institution (WHOI BCO-DMO) |
BCO-DMO Project Info
Project Title | An In-Depth analysis of the subvent biosphere within Okinawa Backarc Basin (IODP 331, Iheya North Hydrothermal Field) sediments |
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Acronym | Subvent_Biosphere_Sediments |
URL | https://www.bco-dmo.org/project/714230 |
Created | September 1, 2017 |
Modified | November 5, 2019 |
Project Description
The Iheya North Hydrothermal Field in the Okinawa Backarc Basin represents an ideal environment in which to investigate the biotic temperature fringe of microbial life at depth because of its subsurface hydrothermal activity within its continental margin-type sediment profile. Geographically, the Okinawa Backarc Basin is situated along a continental margin, which is a sediment profile type commonly sampled and studied across the seafloor (e.g. Peru Margin, Costa Rica Margin, Cascadia Margin). The hydrothermal network within the subsurface here supplies an additional temperature obstacle to microbial life existing in the sediments. In particular, the sediment profile at Site C0014 exhibits a transition from hemipelagic ooze with pumiceous volcaniclastic sediments and low temperature (4°C) to a hydrothermally altered sequence of clays within the top ~10 mbsf of sediment. Temperature measurements indicate a gradient of approximately 3°C/m, which is roughly an order of magnitude greater than continental margin sites (e.g. Cascadia Margin, IODP 311 and Costa Rica Margin, IODP 344), but is more gradual than intense, centimeter-scale gradients from other hot, surface sediments. We have focused on the application of culture-independent, molecular methods to understand taxonomic and functional characteristics through this hydrothermal gradient. Confidence in DNA recovery suggests a microbial biosphere extent of approximately 15 mbsf (55°C).
Results from both 16S rRNA gene surveys and metagenomics analyses suggest a temperature-dependent stratigraphy of taxonomic and functional adaptations between the shallowest and deepest sample horizons. Cosmopolitan marine subsurface bacterial and archaeal taxa are present throughout the top 10 mbsf, whereas, hyperthermophilic heterotrophic as well as thermophilic anaerobic methanotrophic archaea appear in varying local abundances in deeper, hydrothermal clay horizons. “An In-Depth analysis of the subvent biosphere within Okinawa Backarc Basin (IODP 331, Iheya North Hydrothermal Field) sediments” encompasses datasets funded through C-DEBI to investigate the microbial communities in IODP 331 Iheya North Hydrothermal Field sediments. Site C0014 at this field site is 500 m away from the active vent and experiences a 3C/m temperature gradient with depth. In the research grant “Investigating the active microbial populations in near hydrothermal vent sediments” and the Research Exchange grant “Learning new RNA extraction techniques”, we used an RNA-based approach to explore the active microbial community. Upon analysis, the RNA dataset does not appear to reflect the same information as the phylogenetic signals coming from both the 16S rRNA gene as well as the metagenomes. The data indicate significant background noise from the RNA extraction process rather than from an indigenous representation of the subsurface biosphere.
The graduate fellowship “An in-depth analysis of the subvent biosphere within the Okinawa backarc basin Iheya North hydrothermal field” provided an opportunity to bring together metagenomic, 16S rRNA gene amplicon (DNA), and 16S rRNA amplicon datasets from IODP 331 Iheya North Hydrothermal Field sediments to understand the biogeography of this subvent biosphere.
For more information, refer to the following C-DEBI grants/projects.
An in-depth analysis of the subvent biosphere within the Okinawa backarc basin Iheya North hydrothermal field:
https://www.darkenergybiosphere.org/award/an-in-depth-analysis-of-the-subvent-biosphere-within-the-okinawa-backarc-basin-iheya-north-hydrothermal-field/
Investigating the active microbial populations in near hydrothermal vent sediments: https://www.darkenergybiosphere.org/award/investigating-the-active-microbial-populations-in-near-hydrothermal-vent-sediments/
Learn new RNA extraction techniques: https://www.darkenergybiosphere.org/award/learn-new-rna-extraction-techniques/
Data Project Maintainers
Name | Affiliation | Role |
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Christopher H. House | Pennsylvania State University (PSU) | Principal Investigator |
Jennifer F. Biddle | University of Delaware | Co-Principal Investigator |